Redox Enzymes in Tethered Membranes

Redox Enzymes in Tethered Membranes

Lars J.C. Jeukenχ,*, Simon D. Connellχ, Peter J.F. Henderson§, Robert B. Gennis∥, StephenD. Evans†, and Richard J. Bushby‡

χInstitute of Molecular Biophysics, University of Leeds, Leeds LS2 9JT, UK†School of Physics and Astronomy, University of Leeds, Leeds LS2 9JT, UK‡Centre for Self-Organising Molecular Systems, University of Leeds, Leeds LS2 9JT, UK§Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK∥Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA

AbstractAn electrode surface is presented that enables the characterisation of redox-active membraneenzymes in a native-like environment. An ubiquinol oxidase from Escherichia coli, cytochromebo3 (cbo3), has been co-immobilised into tethered bilayer lipid membranes (tBLMs). The tBLM isformed on gold surfaces functionalised with cholesterol tethers which insert into the lower leafletof the membrane. The planar membrane architecture is formed by self assembly ofproteoliposomes and its structure is characterised by surface plasmon resonance (SPR),electrochemical impedance spectroscopy (EIS) and tapping-mode atomic force microscopy (TM-AFM). The functionality of cbo3 is investigated by cyclic voltammetry (CV) and is confirmed bythe catalytic reduction of oxygen. Interfacial electron transfer to cbo3 is mediated by themembrane-localised ubiquinol-8, the physiological electron donor of cbo3. Enzyme coveragesobserved with TM-AFM and CV coincide (2–8.5 fmol·cm−2) indicating that most - if not all - cbo3on the surface is catalytically active and thus retains its integrity during immobilisation.

IntroductionSince the first electrochemistry on a protein was reported almost 30 years ago (cytochromec),1,2 much research has focused on the preparation of ‘protein-friendly’ electrode surfaces.For cytochrome c alone more than a hundred papers have appeared that compare differentelectrode surfaces.3 A key issue has been, and still is, to interface redox-active proteins andenzymes to an electrode surface without changing their structural integrity and functionality.A particularly powerful method has been to immobilise enzymes on the surface, removingthe rate-limiting step of diffusion. Electrochemical studies of adsorbed enzymes allow an in-depth study of the fundamental parameters that typify these enzymes, like catalysis,inhibition and electron flow.4–8

Although many reports have emerged on the electrochemistry of soluble proteins, far lesshas been published on membrane proteins in spite of their central role in processes such asthe mitochondrial electron-transport chain and photosynthesis. Indeed, many redox-activeproteins are located in the mitochondrial, chloroplast or plasma membranes.9 As cytochromec has become an important test case for adapting electrode surfaces to globular proteins, so

*Corresponding author. [email protected], Fax:0044-(0)113-3433900 .

Supporting information available CD spectra of vesicle-reconstituted cbo3 (proteoliposomes); further analysis of the impedancedata shown in Figure 2. This material is available free of charge via the Internet at http://pubs.acs.org.

Europe PMC Funders GroupAuthor ManuscriptJ Am Chem Soc. Author manuscript; available in PMC 2013 February 05.

Published in final edited form as:J Am Chem Soc. 2006 February 8; 128(5): 1711–1716. doi:10.1021/ja056972u.

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http://pubs.acs.org

too has cytochrome c oxidases (CcO) become an important test case for membraneproteins.10–21 Different strategies have been reported each with their own advantages anddrawbacks, such as direct adsorption of detergent solubilised protein on modifiedsurfaces,18–20 the incorporation of CcO in supported membranes21 or hybrid bilayers14–17

and attaching the membrane proteins to the surface followed by the reconstitution of thelipid bilayer.10–12

Our aim is to develop an electrode surface that allows the investigation of a range ofmembrane proteins in a native(-like) environment and is relatively easy to prepare usingself-assembly mechanisms. To fulfil these aims we have used the methodology of tetheredbilayer lipid membranes (tBLM).12,13,22–31 In tBLMs, the bilayer is attached to the surfacevia special chemical anchors, which on one side are bound to the surface and on the otherside insert into a bilayer leaflet (Figure 1). For this purpose lipid derivatives have beensynthesised, which, via a hydrophilic linker, are connected to a group that binds strongly tothe surface. One such molecule we have synthesised and characterised is the cholesterolderivative shown in Figure 1. The thiol group is separated from the cholesterol lipid via anethyleneoxy chain.28–31 Importantly, to form a phospholipid bilayer instead of a monolayerof phospholipids on top of a monolayer of cholesterol tethers, the cholesterol derivatives aremixed with small spacer molecules (6-mercaptohexanol). Self-assembled monolayersprepared from mixed thiol solutions are generally observed to form phase separated domainson the nanoscale, induced by differences in chain length32–36 or chemistry.37–39 Since thetwo thiol compounds used here are very distinct, both in length and chemistry, similar phaseseparation is expected as is schematically shown in Figure 1. This will allow phospholipidsto enter both leaflets of the bilayer and provide space for transmembrane proteins.

Here, we describe an innovation in which the tBLM system is used to immobilise anubiquinol oxidase, cytochrome bo3 (cbo3) from Escherichia coli, on an electrode surface.Like aa3-type cytochrome c oxidases, cbo3 is a member of the heme-copper oxidasefamily40–43 and is a redox-driven proton pump that couples the reduction of water to thegeneration of proton-motive force across the membrane. Cbo3 is a four subunit protein of144 kDa that receives its electrons from the membrane component, ubiquinol-8. Here, wewill show that tBLMs can successfully be formed from proteoliposomes and that cbo3 is co-adsorbed on the surface in a functional and integral form.

MethodsProtein purification and reconstitution

Cytochrome bo3 (cbo3) was purified as previously described from the E. coli strain, GO105/pJRhisA, in which the his-tagged protein is over-expressed,41 except that the detergent n-dodecyl-β-D-maltoside (DDM) was used throughout the purification (1% for solubilisationof the membranes and 0.05% for all subsequent steps in the purification). The purity of thesample was confirmed by SDS-PAGE. The as-isolated protein had a Soret peak at 409 nm -similar to the 408 nm found previously.42 - and the protein concentration was estimatedusing ∈408nm = 188 mM−1cm−1.42 Protein concentrations based on a Schaffner-Weissmanassay44 were about 30% higher than those based on the Soret band. Cbo3 was reconstitutedinto vesicles of an E. coli ‘polar’ lipid extract (Avanti) as described by Carter et al.40 Lipidvesicles of about 100 nm diameter were prepared using a mini extruder (Avanti) to whichoctylglucoside (OG) and protein were added (final concentrations: ~16 mg/ml lipid and ~ 45mM OG). Typically, 1 % protein / lipid (w/w) was used. After 5-10 min. incubation at 4 °Cthe lipid/protein mixture was rapidly diluted (> 100 times) with cold (4 °C) buffer andcentrifuged at 100,000 g for 1 hour to spin down the proteoliposomes. The resuspendedproteoliposomes were used within 2 days. Protein yields after reconstitution weredetermined with a Schaffner-Weissman protein assay and were between 40 and 50% of the

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starting material (for samples with 1 mass percent ratio). Depending on the amount of lipidlost during the preparation this results in proteoliposomes with ~0.5% mass percent of cbo3.CD spectroscopy (Jasco J715 spectropolarimeter) confirmed that cbo3 retained itsconformation during the reconstitution (see supporting information). All experiments wereperformed in buffer (20 mM MOPS pH 7.4, 30 mM Na2SO4) at 20 °C. For the controlexperiments, vesicles were prepared that were treated identically to the proteoliposomesexcept that cbo3 was omitted.

Cytochrome c oxidase (CcO) was purified and reconstituted as previously described fromthe P. denitrificans strain, AO1/pPDWT.45,46 Consequent handling of the proteoliposomeswas identical to that of cbo3.

Electrode materials, microscopy and spectroscopyGold substrates for AFM, SPR and electrochemical experiments were prepared as describedpreviously.47 For the electrochemistry, 150 nm Gold (Advent, 99.99% ) was evaporatedthrough a mask on a 10 nm chromium adhesion layer on cleaned glass microscope slides(Deconex, sonication, methanol) with an Edwards Auto 306 evaporator at < 2 × 10−6 bar.The mask consisted of a circular ‘electrode’ area (⊘ = 5 mm) with a 1 mm link forconnection to the electrochemical equipment. The total area (circular + link) in contact withthe electrolyte solutions is 0.25 ± 0.03 cm2. After evaporation the electrodes were storeduntil used. Before usage the slides were sonicated in DCM, rinsed with MeOH and driedunder N2. Self-assembled monolayers (SAMs) were made by incubating the electrode slidesfor ~ 16 hours in a 2-proponal solution of 0.11 mM cholesterol tether and 0.89 mM 6-mercaptohexanol. The gold slides were thoroughly rinsed with DCM and MeOH, dried withN2 and used within a day. The surface ratio of the different thiols on the surface waschecked for each electrode with impedance spectroscopy, assuming that the capacitance of amixed SAM is equal to the summed capacitance of the pure SAMs times the area occupied.This assumption was previously checked and found to be correct with contact anglemeasurements.47 For the thiol mixture used, the surface area ratio was typically found to be50% /50% cholesterol-tether / 6-mercaptohexanol.

For the electrochemical experiments a glass cell was used that housed a calomel referenceelectrode, Pt counter electrode and glass slide with the gold electrode. The glass cell alsohoused a Clark-type oxygen electrode and had an inlet for argon. The Clark-type oxygenelectrode was positioned < 5 mm from the working electrodes. Two Clark electrodes wereused: one from World Precision Instruments (ISO, resolution ±0.1 mg/L) and one home-built electrode, which was slower in response but had a higher resolution (±0.02 mg/L).Oxygen concentration was typically ‘regulated’ by purging the cell thoroughly with argonuntil anaerobic and then stopping the argon flow while continuously measuring cyclicvoltammograms and monitoring the increase in oxygen concentration in time due to slowoxygen diffusion into the cell via the Argon inlet.

For the AFM and force curve experiments, template stripped gold (TSG) surfaces wereprepared by evaporating 150 nm gold on silicon wafers. The gold surface was then gluedwith EPO-TEK 377 to glass and cured for 120 min at 120° C. After cooling, the slides weredetached from the silicon wafers to expose the TSG surface, which was directly immersed inthe thiol solution (0.11 mM EO3-Cholesterol and 0.89 mM 6-Mercaptohexanol) for onehour. Using electrochemistry it was confirmed that also on TSG surfaces the used thiolmixture results in a 50% /50% cholesterol-tether / 6-mercaptohexanol area ratio. AFMheight images and force curves were recorded under fluid (buffer) at 25° C using aMultimode AFM on a Nanoscope IIIa or IV (Digital Instruments, Veeco Metrology Group,Inc., CA) controller and silicon nitride cantilevers (NP, Veeco Metrology Group). Thecantilevers had a spring constant of approximately 0.18 or 0.52 N/m. The height images

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reported here are raw, unfiltered data obtained by tapping (dynamic) mode at a frequency inthe range 8-9 kHz. Force curves (z-Piezo extension vs cantilever deflection) were recorderunder indentical conditions and converted into true force-distance curves showing force innN as a function of true tip-surface separation in nm.

SPR experiments were performed on prisms evaporated with 50 nm gold as describedpreviously28 and the results were fit by a Levenberg-Marquardt procedure.48,49

Tethered bilayer formationProteoliposomes with cbo3 (or control vesicles) were extruded in a mini extruder (Avanti)using 100 nm nuclepore track-etched membranes (Whatman) and subsequently added to thegold surface in the presence of 10 mM Ca(II). We have shown previously that calcium ionsare necessary to induce the formation of tethered lipid bilayers when lipid extracts are usedthat are derived from E. coli.47 (For a more thorough characterisation of the effects ofcalcium we refer to the work of Richter and Brisson.50) This lipid mixture containsapproximately 20-35% negatively charged phospholipids (phosphatidylglycerol andcardiolipin)51,52 and the Ca(II) might be necessary to overcome the electrostatic repulsionbetween the vesicles. In general it was found that a tethered lipid bilayer was formed within1 hour at 20 °C. After bilayer formation, the surface was rinsed several times with buffersolutions containing 1 mM EDTA to remove Ca(II) ions and remaining vesicles.

ResultsStructural analysis

Tethered lipid bilayers were characterised with surface plasmon resonance (SPR),electrochemical impedance spectroscopy (EIS) and tapping-mode atomic force microscopy(TM-AFM). Figure 2 shows the EIS results in the form of Cole-Cole plots in which thediameter of the half circle is equivalent to the double layer capacitance. It is immediatelyclear that inclusion of cbo3 has almost no effect on the double layer capacitance of thetethered membrane. This indicates that cbo3 does not induce large defects in the tetheredbilayer. The capacitance of the lipid bilayers (0.7–0.8 μF/cm2, see supportive informationfor EIS analysis) is only slightly larger than that of an ideal phospholipid bilayer (0.5 μF/cm2) and similar to that reported previously.29,47,53,54

The increase in bilayer thickness after self-assembly of vesicles without cbo3 wasdetermined with SPR to be 3.8 ± 0.1 nm (n=3). This corresponds with previous studies andis as expected when half the inner lipid layer is occupied by cholesterol tethers.28 Thepresence of cbo3 results in a bilayer that is not significantly thicker (4.3 ± 0.3 nm , n=9, S.D.= 0.9 nm), although we note that a variability between samples was observed. In order tostudy the tethered lipid bilayers further, TM-AFM images of the electrode surface wererecorded.

Figure 3 shows a set of TM-AFM results of tBLMs with and without cbo3. Without cbo3 amostly flat surface (RMS < 0.5 nm) is observed with very infrequently a heightened areadue to an adsorbed vesicle. The presence of a lipid bilayer is confirmed by forcemeasurements (insert Figure 3[Top]), which shows the typical behaviour of a lipid bilayer.55

When cbo3 is present, a large number of heightened areas are observed (Figure 3[Middle]).Most of these are 5 ± 2 nm in height and roughly circular with a diameter of 30-40 nm.Some of the areas are larger with diameters up to 75 nm and a height of ~10 nm. Noheightened areas were observed in contact mode AFM (CM-AFM) at high forces (> 2 nN)confirming they were not due to the underlying gold surface. Furthermore, the raised areaswere damaged or dislocated by the cantilever tip in CM as shown by TM-AFM scansafterwards. On some of the prepared samples with cbo3 adsorbed vesicles were encountered.

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These vesicles were ~20 nm high with diameters > 150 nm. Importantly, these vesicles wereoften removed or dislocated by the tip during TM scanning (see Figure 3[Top] for anexample), indicating that they were loosely bound. We note that adsorbed vesicles weremore frequently observed for samples with cbo3 and the amount of adsorbed vesicles wasvariable between samples.

Functional analysisCbo3 from E. coli is a terminal oxidase and functions as an ubiquinol-8 oxidase.Importantly, the control experiments shown in Figure 4A indicate that the E. coli lipidextract - used throughout this work - still contains ~0.3 mass percent of ubiquinol-8(UQ-8).56 The redox signals in Figure 4A are assigned to UQ-8 based on controlexperiments with EggPC/UQ-10 (not shown) and values reported in literature.47,57 The peakseparation increased at higher scan rates indicating that the electron transfer is kineticallycontrolled.58,59 This is partly due to protonation and deprotonation reactions of UQ-8 asaddition of the proton ionophore, CCCP, sharpens the peaks and reduces the peakseparation. Since UQ-8 is likely to be oxidised or reduced while located at the interfacebetween the gold surface and the lipid bilayer, CCCP is thought to facilitate the protonexchange between this interface and the buffer solution on the other side of the lipid bilayer.The interfacial redox kinetics are also limited by the high co-operativity of the two-electronreaction of the quinones, which is described in more detail by Marchal et al.57 (∣ΔE0∣ ~ 0.6V at pH 7.4). From the peak area it is determined that the average coverage of UQ-8 is ~2pmol·cm−2.

When a tethered bilayer is formed with vesicles containing cbo3 a sigmoidal wave isobserved starting at the same potential as the reduction of UQ-8 (Figure 4B) indicating twoimportant points: (1) cbo3 located in the tethered membrane is at least partly active; and (2)electrons are transferred from the gold electrode to cbo3 via UQ-8. Varying the substrate(oxygen) concentration in the electrochemical cell changed the magnitude of the catalyticwave as expected (Figure 4C). The apparent Km of cbo3 for oxygen in the electrochemicalsetup was found to vary between experiments, which might reflect the instability of theClark-type oxygen electrodes or variable O2 diffusion gradients. However, the

was always < 3 μM. Addition of extra UQ-10 to the membrane did not significantly increaseenzyme activity, indicating that the amount of UQ-8 adsorbed on the surface is >>Km.Addition of 1 mM CN− completely inhibitted cbo3 and, consequently, UQ-8 oxidation/reduction peaks were observed that are similar to those shown in Figure 4A.

DiscussionSurface structure

The results demonstrate that it is possible to incorporate the membrane protein, cbo3, in atBLM using self-assembly methods. Impedance spectroscopy indicates that the bilayercapacitance is identical to that of membranes without cbo3, suggesting that self-assembly ofcbo3 does not induce large defects in the membrane.

TM-AFM results show that cbo3 is randomly distributed across the surface. The height (Zdirection) is consistent with that of cbo3, which is ~10 nm high (see Figure 543) and istherefore expected to extend about 5 nm above the surrounding lipid bilayer. In contrast, thesize in the xy-direction is much larger (30-75 nm) than the size of cbo3. This could be due toelevation of the lipid bilayer by the protein since the space between the membrane and the

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surface in the tBLM system is not sufficient to accommodate the extramembranous domainsof cbo3. However, it is also likely that the size is artificially enlarged by the geometry of thecantilever tip.60 We cannot exclude the possibility that multiple proteins are located withineach heightened area or that some of the larger areas represent semi-fused vesicles ordefects.

A reconsitution procedure very similar to the one used in this work has been shown give anunidirectional orientation of cbo3 in the vesicles.61 However, in order to acquire goodquality tBLMs the reconstituted vesicles had to be extruded through a 100 nm track-etchedmembrane, which might randomise the protein orientation. Regretfully, due to the lowresolution, the orientation of cbo3 could not be unambiguously determined with AFM.

Enzyme activityThe tethered bilayers are formed by self-assembly of vesicles with ~0.5 mass percent ofcbo3 (see Methods). If it is assumed that all cbo3 assembles when the tethered bilayer isformed and that half the surface is occupied by cholesterol tethers, cbo3 coverage isexpected to be 8 fmol·cm−2.62 This is confirmed by the AFM results. Particle analysis of thedata shown in Figure 3 (middle) gives 150-175 molecules in a 2 × 2 μm area, correspondingto 6 - 7.5 fmol·cm−2. Analysis over a larger set of data shows that the coverage variesbetween 2 and 7.5 fmol·cm−2. Importantly, the same enzyme coverage is indicated by theelectrochemical results. Using the published maximum activity of cbo3 (Vmax in solution is730 e−·s−1)41 and the catalytic activity on the electrode (400 - 600 nA·cm−2, Figure 4B) theactive enzyme coverage can be estimated at 5.6 and 8.5 fmol·cm−2. Analysis of moreelectrodes shows that the active enzyme coverage varies between 3 - 8.5 fmol·cm−2. Theseestimates confirm that a large part - if not all - of the cbo3 on the surface is active. In thisrespect it is important to note that maximum enzyme activity is achieved at O2 levels below10 μM (< 0.3 ppm), which is well below the level at which gold-catalysed oxygen reduction(i.e., the background) becomes a significant part of the voltammogram.

Comparison with other systemsTo our knowledge, five groups have reported on the immobilisation of cytochrome c oxidase(CcO) on electrode surfaces.10–21 Of these, slow, but direct electron transfer from theelectrode to the immobilised protein has been described by Hawkridge et al.,14–17 Dong etal.19,20 and Tollin et al.21 However, as is also noted by Dutton et al.,18 in none of thesesystems did the interfacial electron transfer induce catalytic oxygen reduction by CcO. Incontrast, sigmoidal reduction waves typifying catalytic activity have been observed byNaumann et al.,10 Dutton et al.18 and in the work described here. Both Naumann et al. andDutton et al. used the physiological redox partner, cytochrome c, to mediate electron transferfrom the electrode to the immobilised enzyme.10,18 Our work corresponds with theseobservations since the physiological electron donor, UQ-8, was needed to mediate electrontransfer between the electrode and cbo3. Furthermore, no catalytic activity was observedwhen tBLMs were formed with CcO from P. denitrificans nor was direct interfacial electrontransfer to CcO detected. To observe clearly a sigmoidal wave in the voltammograms, slowscan speeds are required (< 2 mV·s−1) so that the signals of the mediators (which depend onscan rate) will be small relative to the catalytic currents. The catalytic currents obtained afterbaseline subtraction are ~50,18 ~13310 and ~500 (this work) nA·cm−2. These relatively smallcatalytic currents (compared to most other voltammetric results of globular enzymes) eitherreflect low enzyme coverage (as shown for here for cbo3), partially deactivated enzyme orlimiting activity of the electron mediator. Further comparison between the work describedhere and previous work on CcO is difficult since the electron transfer between cytochome cand CcO is very different in nature than between ubiquinol and cbo3.

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Concluding remarksWe have shown that redox-active membrane proteins can conveniently be co-immobilised intBLM systems using self-assembly methodology. For enzymes that interact with the quinonepool, electrochemical interaction is possible using quinone as the natural mediator. It istherefore expected that this technique is readily applicable to a range of enzyme systems.The tBLMs are prepared from proteoliposomes which require purified proteins andreconstitution into vesicles. Although these techniques are now used routinely, they are stillfar from trivial for many proteins. We are, therefore, currently investigating whether similartBLM systems can be prepared with total membrane extracts, omitting the purification andreconstitution steps. In this respect we note that recently the group of Bourdillon hassucceeded in preparing tBLM structures from inner mitochrial membranes.63

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsCcO was purified from a P. denitrificans strain, AO1/pPDWT, which was generously provided by Dr. A. Puustinenand Prof. M. Wikström (University of Helsinki). This research was supported by an UK BBSRC David Phillipsfellowship to LJ, (24/JF/19090), and by BBSRC and EPSRC grants to PJFH, SDE and RJB.

Abbreviations

Cbo3 Cytochrome bo3

CcO Cytochrome c Oxidase

CV Cyclic Voltammetry

EIS Electrochemical Impedance Spectroscopy

TM-AFM Tapping-Mode Atomic Force Microscopy

SPR Surface Plasmon Resonance

UQ UbiQuinol

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50. Richter RP, Brisson AR. Biophys. J. 2005; 88:3422–3433. [PubMed: 15731391]

51. Wilkinson, SG. Gram-negative bacteria. In: Ratlegde, C.; Wilkinson, SG., editors. MicrobialLipids. Vol. Vol. 1. Academic Press; London: 1988.

52. See also www.avantilipids.com

53. Naumann R, Schiller SM, Giess F, Grohe B, Hartman KB, Karcher I, Koper I, Lubben J, VasilevK, Knoll W. Langmuir. 2003; 19:5435–5443.

54. Terrettaz S, Mayer M, Vogel H. Langmuir. 2003; 19:5567–5569.

55. Dufrêne YF, Boland T, Schneider JW, Barger WR, Lee GU. Faraday Discuss. 1998:79–94.[PubMed: 10822601]

56. The concentration of UQ-8 in the tBLM on the surface is approximately 2 pmol·cm−2. Using thebilayer thickness of 3.8 nm (this is without the cholesterol tethers as determined with SPR) andassuming an average weight density of the lipid bilayer of 1.2 g/mL, the concentration of UQ-8 is0.3 mass percent.

57. Marchal D, Boireau W, Laval JM, Bourdillon C, Moiroux J. J. Electroanal. Chem. 1998; 451:139–144.

58. Laviron, E. Voltammetric Methods for the Study of Adsorbed Species. In: Bard, AJ., editor.Electroanalytical Chemistry. Vol. Vol. 12. Marcel Dekker; New York: 1982.

59. Hirst J, Armstrong FA. Anal. Chem. 1998; 70:5062–5071. [PubMed: 9852788]

60. The lipid are expected to modify the AFM probe when imaging bilayer (this has also beenobserved by other groups performing AFM on bilayers, see ref.50 and references therein). A tipwith radius 10 nm would have its asperities submerged and smoothed out and broadens the tipradius to 13 nm. If 5 nm of cbo3 is sticking out of the bilayer, then an AFM tip with a radius of 13nm would result in an image of a hemispherical blob ≈30 nm in diameter and losing the fine detail.

61. Verkhovskaya ML, GarciaHorsman A, Puustinen A, Rigaud JL, Morgan JE, Verkhovsky MI,Wikstrom M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94:10128–10131. [PubMed: 9294174]

62. To estimate the coverage in fmol·cm−2 it is assumed that the surface area/mass ratio of the lipidbilayer is 0.05 Å2·Da−1. To our knowledge, this value for the total lipid extract of E. coli has notbeen determined, but the mean area of most phospholipids lies between 55 and 85 Å2 while theirmolecular weight lies between 700 and 800 Da. The surface area/mass ratio of cbo3 can beestimated at 0.02 Å2·Da−1 (see Figure 5, MW of cbo3 is 144 kDa). Thus when 0.5% of the mass ofthe proteoliposomes is cbo3, 0.2% of the surface is occupied by cbo3. 2×10−4/3×1017 m2 is7×1012 cbo3 molecules·m−2, which is 10 fmol·cm−2. Since half the surface is occupied bycholesterol tethers, a quarter of the total tBLM will be filled by the tether molecules, lowering thecbo3 coverage on the surface by a quarter.

63. Elie-Caille C, Fliniaux O, Pantigny J, Maziere JC, Bourdillon C. Langmuir. 2005; 21:4661–4668.[PubMed: 16032886]

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Figure 1.Chemical structures of 6-mercaptohexanol (spacer) and the cholesterol tether molecule usedto form tBLMs. In the middle is a schematic representation of a tethered lipid bilayer formedon a mixed self-assembled monolayer of tether and spacer molecules.

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Figure 2.Cole-Cole plots before and after formation of a tBLM measured at 0 V vs. SCE. Top:Control (without cbo3). Bottom: With cbo3. The lines represent fits as is described inSupporting Information.

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Figure 3.TM-AFM images of tBLMs on template stripped gold. Top: Control experiment withoutcbo3. For illustration purposes we show an image with an adsorbed vesicle, although thesewere very infrequently encountered in control experiments. The arrow shows a singlevesicle being removed during the scan (scan direction is from bottom to top). Insert: Typicalforce curve of tBLM layer (solid line, tip approaching surface; dashed line, tip recedingfrom surface). Analysis of this set of force curves indicated an average penetration depth ofthe bilayer of 4.5 ± 0.1 nm (S.D. = 0.9 nm) and a ‘piercing force’ of 1.50 ± 0.02 nN (S.D. =0.4 nN). Middle: tBLM with cbo3. Bottom: Height profile of the middle image indicated bythe line.

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Figure 4.Cyclic voltammograms (CVs) of tBLM systems (electrode area = 0.25 cm2). (A) Controlsample at 10 mV·s−1. tBLMs (without cbo3) before and after addition of 10 μg/ml carbonylcyanide 3-chlorophenylhydrazone (CCCP). Baseline subtracted signals are shown with anoffset and 4 times enlarged. (B) CVs at 1 mV·s−1 before and after formation of a tBLM withcbo3. O2 in both CVs is ~14 μM. (C) Baseline corrected CVs at 1 mV·s−1 of tBLM withcbo3 at different O2 concentrations, (1) ~0, (2) ~0.2, (3) ~0.9 and (4) ~14 μM. CV (4) isderived from the same data shown in (B).

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Figure 5.Space-filling representation of the structure of cytochrome bo .43 A relatively largeextramembranous domain of subunit II of cbo3 is indicated in the structure on left.

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Redox Enzymes in Tethered Membranes

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